Diphenylhydantoin derivatives

ABSTRACT

Reagents for use in binding assays, particularly immuno-assays, to determine diphenylhydantoin and for preparing reagents employed in such assays, including β-galactosyl-umbelliferone-diphenylhydantoin conjugates, diphenylhydantoin immunogens, and N 1 , N 3  and ο-phenyl derivatives of diphenylhydantoin for preparing same. N 1  and N 3  -ω-aminoalkyl and ο-(ω-aminoalkoxy)-phenyl derivatives are prepared and coupled by amide linkage to a β-galactosyl-umbelliferone derivative to form labeled conjugates useful in homogeneous or heterogeneous binding assays. N 1  and N 3  -ω-carboxyalkyl and ο-(ω-carboxyalkoxy)-phenyl derivatives are prepared and coupled by amide linkage to an immunogenic polyamino acid to form immunogen conjugates against which diphenylhydantoin-specific antibodies can be raised.

This is a division, of application Ser. No. 899,844, filed Apr. 25,1978.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel diphenylhydantoin derivatives pertainingto binding assays for detecting diphenylhydantoin and its salt forms inliquid media, such as serum, saliva, and cerebrospinal fluid. Suchderivatives include labeled diphenylhydantoin conjugates directly usedin carrying out such assays. Also described are novel immunogenconjugates for preparing diphenylhydantoin-specific antibodies accordingto conventional techniques. Further, there are described novelintermediates useful in the synthesis of such labeled conjugates andsuch immunogen conjugates.

Diphenylhydantoin (5,5-diphenyl-2,4-imidazolidiendione), also known bythe generic name phenyltoin and by various trademarks includingDilantin, is an anti-convulsant drug useful in the management ofepilepsy, having the formula: ##STR1## wherein φ represents phenyl [cf.The Merck Index, 9th edition, p. 952 (1976)].

Like most anti-convulsants, diphenylhydantoin possesses a lowtherapeutic index and has clearly separable ranges of drug concentrationin blood in which it is ineffective, therapeutic, or toxic,respectively. At sufficiently high concentration, the drug ispotentially toxic and is eliminated from the body at a rate which canvary within a wide range from individual to individual so that the samedose can yield an ineffective, a therapeutic, or a toxic concentrationdepending on the subject. Administration of usual doses ofdiphenylhydantoin produces serum concentrations from 5 up to 50micrograms/milliliter (μg/ml) due to differences in the rate of hepaticmetabolism of the drug. However, in almost all patients, the therapeuticrange of serum concentration lies between 10 and 20 μg/ml whereas toxicsigns of nystagmus, ataxia and mental changes almost invariably appearat blood levels over 20 μg/ml. With usual doses of diphenylhydantoin,some patients will have serum concentrations outside the therapeuticrange, indicating the need for dosage adjustment.

2. Brief Description of the Prior Art

Over the years, many varied assay techniques have evolved for themonitoring of diphenylhydantoin levels in serum, includingspectrophotometry, thin-layer chromatography, high-pressure liquidchromatography, and immunological methods. The state-of-the-artimmunoassay techniques comprise the radioimmunoassay methods of Tigelaaret al, Clin. Chim. Acta 43:231-241(1973) and Cook et al as described inQuantitative Analytic Studies in Epilepsy, ed. Kellaway et al, RavenPress (New York, 1976), pp. 39-58; and the enzyme immunoassay techniquedescribed in U.S. Pat. Nos. 3,817,837 and 3,905,871.

A specific binding assay for detecting ligands, including drugs such asdiphenylhydantoin, employing an enzyme-cleavableβ-galactosyl-umbelliferone residue as label is described in pending U.S.patent application Ser. No. 886,094, filed Mar. 13, 1978, assigned tothe instant assignee.

Methods of synthesizing diphenylhydantoin immunogen conjugates and usingsame to obtain specific antibodies are described by Tigelaar et al,supra and Cook et al, Res. Commun. Chem. Pathol. and Pharmacol.5:767-774(1973) and in U.S. Pat. No. 3,995,021 and French Pat. No.2,276,317.

SUMMARY OF THE INVENTION

Novel labeled diphenylhydantoin conjugates have been devised for use inbinding assays having the general formula: ##STR2## wherein n=2 through6, and preferably is 4; ##STR3## Theseβ-galactosyl-umbelliferone-diphenylhydantoin conjugates are prepared byreaction of a mixed anhydride (fromβ-galactosyl-umbelliferone-3-carboxylic acid and an alkyl chloroformate)with N¹ and N³ -ω-aminoalkyl and ο-(ω-aminoalkoxy)-phenyl-(comprising 2through 6, preferably 4, methylene groups)-diphenylhydantoinderivatives.

The novel immunogen conjugates consist of the N¹ -conjugated compound ofthe formula: ##STR4## and the ο-phenyl-conjugated compound of theformula: ##STR5## wherein, for both formulae, φ is phenyl; PAA is animmunogenic polyamino acid, preferably an albumin, bonded through anamide linkage; n=2 through 6, and preferably 4; and p=1 through 50,preferably 5 through 25. These immunogen conjugates are prepared byreaction of N¹ substituted ω-carboxyalkyl and ο-(ω-carboxyalkoxy)-phenylsubstituted (comprising 2 through 6, preferably 4, methylene groups)diphenylhydantoin derivatives with the polyamino acid under conditionsfavorable to the formation of amide linkages, such as in the presence ofa carbodiimide in acidic solution.

The polyamino acid may be naturally occurring or synthetic and isusually an immunogenic polypeptide or protein. The polyamino acid maycomprise constituents in addition to amino acids and will usually be ofmolecular weight between 5,000 and 1,000,000; preferably between 15,000and 500,000, and more usually between 30,000 and 200,000. For the mostpart, proteins taken from one species will be immunogenic whenintroduced to the blood stream of another species. Particularly usefulproteins are albumins, globulins, enzymes, hemocyanins, albuminoids,glutelins, proteins having significant non-proteinaceous constituents,e.g., glycoproteins, and the like. The albumins and globulins ofmolecular weight between 30,000 and 200,000 are particularly preferred.

Preparation of specific antibodies using the present immunogenconjugates may follow any conventional technique. Numerous texts areavailable describing the fundamental aspects of inducing antibodyformation, for example reference may be made to C. W. Parker,Radioimmunoassay of Biologically Active Compounds, Prentice-Hall(Englewood Cliffs, New Jersey, USA, 1976). In the usual case, a hostaminal such as a rabbit or goat is injected at one or more of a varietyof sites with the immunogen conjugate, normally in mixture with anadjuvant. Further injections are made at the same or different site orsites at regular or irregular intervals thereafter with bleedings beingtaken to assess antibody titer and its rate of increase until it isdetermined that optimal titer has been reached. The host animal issacrificed by exsanguination to yield a suitable volume of specificantiserum. Where desirable, purification steps may be taken to removeundesired material such as non-specific antibodies before the antiserumis considered suitable for use in performing actual assays.

1. N¹ -Derivatives 1-I. PREPARATION OF THE LABELED CONJUGATE

The N¹ -conjugated β-galactosyl-umbelliferone-diphenylhydantoin labeledcompounds are prepared according to the reaction scheme shown inTable 1. This synthetic route is exemplified by the following method ofpreparingN-[4-(5,5-diphenylhydantoinyl-1)-butyl]-7-β-galactosylcoumarin-3-carboxamide(7). To follow this synthesis in Table 1, n equals 4.

5,5-Diphenyl-1-[4-(N-phthalimido)-butyl]-hydantoin (2)

Under an argon atmosphere, 2.4 g (0.05 mol) of sodium hydride (50%dispersion in mineral oil) was placed in a dry, 1 liter, 3-necked roundbottom flask equipped with a mechanical stirrer and thermometer. Thesodium hydride was rinsed with 250 milliliters (ml) of dry hexane. Tothe washed material was added a solution of 16.2 grams (g) (0.05 mol) of3-carbethoxy-5,5-diphenylhydantoin (1) [L. Call, Monat. Chemie 101, 228(1970)] in 250 ml of dry dimethylformamide (DMF). When gas evolutionceased (about 15 minutes), a solution of 15.5 g (0.055 mol) ofN-(4-bromobutyl)-phthalimide was added and the reaction stirredovernight at room temperature. The resulting mixture was then combinedwith 5 ml of glacial acetic acid and evaporated under reduced pressureto give an oil. The oil was adsorbed onto 150 g of silica gel 60 (E.Merck Co., Darmstadt, West Germany) and used to top a column of 1000 gof silica gel 60 made up in 17:3 (v:v) toluene:ethanol. 20 ml fractionswere collected.

                                      TABLE 1                                     __________________________________________________________________________     ##STR6##                                                                      ##STR7##                                                                     __________________________________________________________________________

Fractions 141 to 250 were combined and evaporated. The residue wasrecrystallized from toluene-ethanol to give 6 g of the desiredphthalimido-hydantoin (2) as fine white needles, mp 219° C.

Analysis: Calculated for C₂₇ H₂₃ N₃ O₄ : C, 71.51; H, 5.11; N, 9.27.Found: C, 71.67; H, 5.29; N, 8.96.

NMR Spectrum (d₆ DMSO): δ 0.7 (m, 2H), 1.3 (m, 2H), 3.3 (m, 4H), 7.2 (s,10H), 7.9 (1s, 4H).

1-(4-Aminobutyl)-5,5-diphenylhydantoin (3)

A mixture of 1 g (2.2 mmole) of5,5-diphenyl-1-[4-(N-phthalimido)-butyl]-hydantoin (2), 25 ml ofabsolute ethanol, and 5 ml of 88% hydrazine was refluxed under an argonatmosphere for 1 hour. After complete dissolution, a heavy whiteprecipitate formed. When cool, the excess solvent was removed under highvacuum to leave a white solid residue. The residue was heated for 30minutes in 100 ml of 1 N hydrochloric acid, then cooled. The insolublematerial was filtered and discarded, and the filtrate neutralized withaqueous sodium bicarbonate solution. The precipitate was separated andrecrystallized from pyridine to give 520 milligrams (mg) of the desiredamino-hydantoin (3) as very fine white crystals, mp 254°-256° C.

Analysis: Calculated for C₁₉ H₂₁ N₃ O₂ : C, 70.56; H, 6.55; N, 12.99.Found: C, 70.11; H, 6.53; N, 12.90.

NMR Spectrum (D₂ O-NaOD): δ 0.9 (m, 4H), 2.1 (m, 2H), 3.2 (m, 2H), 7.2(s, 10H).

Alternate Preparation of 1-(4-aminobutyl)-5,5-diphenyl hydantoin (3)

Under an argon atmosphere, 4.32 g (0.09 mol) of sodium hydride (50%dispersion in mineral oil) was placed in a dry, 1 liter, 3-necked roundbottom flask equipped with a mechanical stirrer and thermometer. Thesodium hydride was rinsed with 250 ml of dry hexane. To the washedmaterial was added 29 g (0.09 mol) of 3-carbethoxy-5,5-diphenylhydantoin(1) [L. Call, Monat. Chem. 101:228(1970)] dissolved in 250 ml of drydimethylformamide (DMF). After stirring for one hour, hydrogen gasevolution ceased. Then 20.9 g (0.1 mol) of ethyl-5-bromovalerate wasadded and the reaction stirred overnight at room temperature. The DMFwas removed under high vacuum and the residue partitioned between 500 mlof ether and 300 ml of water (H₂ O). The ether phase was separated,washed with saturated sodium chloride solution, dried over anhydrousmagnesium sulfate, filtered, and evaporated to give an oil. This oil wasstirred at 5° C. for 3 hours in 500 ml of 2 N sodium hydroxide.Acidification with dilute hydrochloric acid precipitated an oil that wascrystallized from ether and recrystallized from aqueous methanol to give5 g of 1-(4-carboxybutyl)-5,5-diphenylhydantoin (4) as white crystals,mp 207° C.

Analysis: Calculated for C₂₀ H₂₀ N₂ O₄ : C, 68.17; H, 5.72; N, 7.95.Found: C, 68.14; H, 5.88; N, 7.73.

NMR Spectrum (C₅ D₅ N): δ 0.8 (m, 4H), 1.65 (t, 2H, J=8 Hz), 2.9 (t, 2H,J=8 Hz), 6.8 (m, 10H).

The mother liquors were combined and chromatographed on 1000 g of silicagel 60, eluting with 17:3 (v:v) benzene: methanol. Fractions 25 to 75were combined, evaporated, and twice recrystallized from aqueousmethanol to give an additional 2.5 g of the acid (4), mp 207° C.

A mixture of 6.4 g (0.018 mol) of1-(4-carboxybutyl)-5,5-diphenylhydantoin (4), 50 ml of dry DMF, and 6 mlof triethylamine was cooled to 0° C. under argon while stirring. To thiswas added 3.2 ml (4.34 g, 0.04 mol) of ethyl chloroformate. After 1 hourat 0° C., the reaction was filtered to remove the precipitate oftriethylamine hydrochloride. The filtrate was cooled to 0° C. andcombined with 100 ml of DMF and 3.5 g (0.054 mol) of sodium azide in 40ml of H₂ O. After 2 hours at this temperature, the reaction was dilutedwith 2 liters of H₂ O and extracted with three 500 ml portions of ether.The ether extracts were combined and evaporated at 20° C. under reducedpressure. The residue amounted to 7 g of an off-white oil. The oil wastaken up in 150 ml of absolute ethanol and refluxed for 2 hours. Whencool, the resulting mixture was evaporated to give a clear white oil.Recrystallization from aqueous ethanol gave 1.6 g of fine crystals, mp174° C., of 1-[4-(N-carbethoxy)-aminobutyl]-5,5-diphenylhydantoin (5).

Analysis: Calculated for C₂₂ H₂₅ N₃ O₄ : C, 66.81; H, 6.37; N, 10.63.Found: C, 66.20; H, 6.45; N, 10.28.

Mass Spectrum (70eV) m/e: 395 [M⁺ ]. 349 [M⁺ minus OC₂ H₅ ], 322 [M⁺minus COOC₂ H₅ ], 306 [M⁺ minus NHCOOC₂ H₅ ].

A mixture of 1.5 g (4 mmole) of1-[4-(N-carbethoxy)-aminobutyl]-5,5-diphenylhydantoin (5) and 50 ml of 1N sodium hydroxide was refluxed for 16 hours, then cooled andneutralized with carbon dioxide. Carbon dioxide was removed from thesolution under reduced pressure and the precipitate filtered andrecrystallized from dimethylsulfoxide to give 1.1 g of theaminohydantoin (3) as fine white needles, mp 254°-256° C.

Analysis: Calculated for C₁₉ H₂₁ N₃ O₂ : C, 70.56; H, 6.55; N, 12.99.Found: C, 69.19; H, 6.57; N, 12.23.

Mass Spectrum (70eV) m/e: 323 [M⁺ ], 324 [MH⁺ ], 254 [(C₆ H₅)₂ CHNHCH₂CH₂ CH₂ CH₂ NH₂ ⁺ ].

N-[4-(5,5-Diphenylhydantoinyl-1)-butyl]-7-β-galactosylcoumarin-3-carboxamide(7)

The potassium salt of the β-galactoside of 3-carboxy-7-hydroxycoumarin[Burd et al, Clin. Chem 23, 1402 (1977)] (808 mg, 2 mmol) was suspendedin 10 ml of dry DMF and cooled to 0° C. while stirring under an argonatmosphere. To this was added 216 mg (2 mmol) of ethyl chloroformate andthe reaction stirred for 3 hours to form the mixed anhydride (6). Then969 mg (3 mmol) of 1-(4-aminobutyl)-5,5-diphenylhydantoin (3), 610 mg (5mmol) of 4-dimethylaminopyridine, 10 ml of dry pyridine, and 10 ml ofDMF were added. Stirring was continued at room temperature overnight.

To the reaction mixture was added 7 g of silica gel 60 and the solventremoved under reduced pressure. The impregnated silica gel was placedatop a column of 200 g of silica gel made up in 4:2:1 (v:v:v)n-butanol:methanol:H₂ O. Elution was with the same solvent and 20 mlfractions were collected. Fractions 34-49 were combined and evaporatedto give 550 mg of crude product which was rechromatographed on 200 g ofsilica gel 60 eluting with a gradient of ethyl acetate-ethanol. Thisgave 300 mg of an amorphous off-white solid which was taken up in 10 mlof methanol and chromatographed on a 45 cm by 3.2 cm column of SephadexLH-20 (Pharmacia Fine Chemicals, Piscataway, New Jersey, USA) elutingwith methanol. Seven ml fractions were collected. Fractions 36 to 44were combined and evaporated to give 200 mg of a clear, faintly yellowglassy solid of the labeled conjugate (7).

Analysis: Calculated for C₃₅ H₃₅ N₃ O₁₁ : C, 62.40; H, 5.24; N, 6.24.Found: C, 61.74; H, 5.05; N, 6.17.

[α]_(D) =-35.79° (c 1.0, methanol).

The above-described synthesis of the N¹ -labeled conjugate (7), n=4 ,can be modified to yield labeled conjugates wherein n=2 through 6 byreplacing N-(4-bromobutyl)-phthalimide in the described synthesis of thephthalamido-hydantoin (2) with the appropriateN-(ω-bromoalkyl)-phthalimide as follows:

    ______________________________________                                        n                   alkylene                                                  ______________________________________                                        2                   ethylene                                                  3                   propylene                                                 5                   pentylene                                                 6                   hexylene                                                  ______________________________________                                    

1-II. PREPARATION OF THE IMMUNOGEN CONJUGATE

This synthesis is shown schematically in Table 2 and is exemplified forn=4 as follows:

26.4 mg (75 μmol) of 1-(4-carboxybutyl)-5,5-diphenylhydantoin (4)[prepared from 3-carbethoxy-5,5-diphenylhydantoin (1) as described in1-I] was dissolved in 0.75 ml dioxane. The solution was cooled to 5° to10° C. in an ice water bath and 17.5 μl (14.5 mg, 75 μmol) of ethylchloroformate was added and mixed. The solution was allowed to react for15 minutes at 5° to 10° C. before adding it to the protein solution. Theprotein solution was prepared by dissolving 125 mg (2.08 μmol) of bovineserum albumin (BSA, represented in Table 2 as PAA, Research ProductsDivision of Miles Laboratories, Inc., Elkhart, Indiana USA) in 3.25 mlH₂ O containing 125 μl of 1 N sodium hydroxide. While vortex mixing,3.25 ml dioxane was slowly added to the alkaline BSA solution. The BSAsolution was then placed in an ice bath and the activateddiphenylhydantoin derivative added. After 2 hours, the reaction mixturewas brought to room temperature. 5 drops of 1 N sodium hydroxide wasadded to clear the cloudy solution (the pH rose to above 9).

                  TABLE 2                                                         ______________________________________                                         ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                

The reaction mixture was chromatographed with 50 mM ammonium formatethrough a 2.5×90 cm column of G-10 Sephadex. The immunogen conjugate (8)eluted in the column void volume. Spectral analysis of the productindicated 18.6 moles of diphenylhydantoin per mole of BSA.

The above-described synthesis of the N¹ -immunogen conjugate (8), n=4,can be modified to yield conjugates wherein n=2 through 6 by replacingethyl-5-bromovalerate in the described synthesis of the acid (4) asfollows:

    ______________________________________                                        n               starting material                                             ______________________________________                                        2               ethyl-3-bromopropionate                                       3               ethyl-4-bromobutyrate                                         5               ethyl-6-bromocaproate                                         6               ethyl-7-bromoheptanoate                                       ______________________________________                                    

1-III BINDING ASSAY FOR DIPHENYLHYDANTOIN

A. Reagents

1. Antiserum--Rabbits were immunized with the N¹ -conjugated immunogenprepared according to 1-II above.

2. Enzyme--Escherichia coli grade IV β-galactosidase was used (SigmaChemical Co., St. Louis, Missouri, U.S.A.). The enzyme preparation usedhad a specific activity of 666 units per mg of protein. One unit ofactivity was defined as that amount which hydrolyzed 1.0 μmol ofο-nitrophenyl-β-D-galactoside per minute at pH 7.2 and 37° C.

3. Buffer--Bicine buffer [N,N-bis-(2-hydroxyethyl)-glycine, NutritionalBiochemicals Corp., Cleveland, Ohio, USA] was used at pH 8.2 and 50mmolar at 25° C.

4. Diphenylhydantoin Standards--25 mg of diphenylhydantoin was dissolvedin 10 ml of dimethylsulfoxide (DMSO) to give a 2500 μg/ml solution. By1:9 serial dilution with DMSO, 10 ml volumes of four additionalstandards were prepared to concentrations of 250, 25, 2.5, and 0.25μg/ml, respectively. A negative standard comprising 10 ml of DMSOcontaining no diphenylhydantoin was also used.

B. Apparatus

Fluorescence was measured with an Aminco-Bowman Spectrophotofluorometer(American Instrument Co., Silver Springs, Maryland, U.S.A.). Excitationand emission wavelengths were set at 400 and 453 nm, respectively.Reaction rates were monitored on a strip-chart recorder connected to thefluorometer. Reaction rates are presented herein as the change instrip-chart units (fluorescence units) per minute. All fluorescencemeasurements were conducted at 25° C.

C. Assay Procedure

3.0 ml aliquots of a reagent were prepared in 50 mM Bicine buffer (pH8.2) in a cuvette to contain 0.0157 units/ml of β-galactosidase and anamount of antiserum sufficient to decrease the reaction rate to about15% of that observed in the absence of antibody. To separate aliquots ofthe reagent were added 10 μl of the various standard diphenylhydantoinsolutions. After mixing, 10 μl of a 12 μM aqueous solution of the N¹-labeled conjugate (prepared as described in 1-I above) in 0.2%(volume:volume) Tween-20 surfactant (a polyoxyethylene derivative offatty acid partial esters of sorbitol anhydride from J. T. Baker,Phillipsburg, New Jersey, USA) was added to each cuvette and the rate ofincrease of fluorescence monitored for 2 to 3 minutes. A control wasalso run following the same procedure except that the initial reagentcontained no antiserum.

D. Results

The response to the various standards was expressed for each cuvette aspercent of competition defined as: ##EQU1##

The results are given in the following table:

    ______________________________________                                        diphenylhydantoin                                                             concentration in      percent of                                              standard (μg/ml)   competition                                             ______________________________________                                        2500                  84.2                                                    250                   61.3                                                    25                    32.9                                                    2.5                   9.3                                                     0.25                  2.0                                                     ______________________________________                                    

The results demonstrate that the labeled conjugate and the antiserumprepared using the immunogen conjugate are useful in an assay fordiphenylhydantoin.

2. N³ -Derivatives 2-I. PREPARATION OF THE LABELED CONJUGATE

The N³ -conjugated β-galactosyl-umbelliferone-diphenylhydantoin labeledcompounds are prepared according to the reaction scheme shown in Table3. This synthetic route is exemplified by the following method ofpreparingN-[4-(5,5-diphenylhydantoinyl-3)-butyl]-7-β-galactosylcoumarin-3-carboxamide(12). To follow this synthesis in Table 3, n equals 4.

3-(4-Aminobutyl)-5,5-diphenylhydantoin (10)

A mixture of 9 g (0.025 mol) of 3-(4-carboxybutyl)-5,5-diphenylhydantoin(9) [Cook et al, Res. Comm. in Chem. Path. Pharmacol. 5:767(1973)], 3.6ml of triethylamine, and 70 ml of dry DMF were cooled to -5° C. whilestirring under argon. To this mixture was added 2.77 g (0.025 mol) ofethyl chloroformate. After 90 minutes the reaction was filtered toremove precipitated triethylamine hydrochloride. An additional 100 ml ofDMF was added and the temperature of the solution adjusted to 5° C. Asolution of 5.1 g (0.078 mol) of sodium azide in 40 ml of H₂ O was addedat such a rate as to keep the temperature between 5 and 7° C. After 2hours stirring at this temperature, the reaction was diluted with 1.2liters of H₂ O and extracted with seven 300 ml portions 1:1 (v:v)ether:pentane. The combined organic extracts were dried over anhydrousmagnesium sulfate, filtered and evaporated to give 6.5 g of a gummyresidue which was taken up in 150 ml of absolute ethanol and refluxedfor 4 hours.

                                      TABLE 3                                     __________________________________________________________________________     ##STR11##                                                                    __________________________________________________________________________

Evaporation of the ethanol gave an oil that was dissolved in 100 ml ofdioxane and 100 ml of 6 N hydrochloric acid and refluxed overnight. Whencool, the reaction mixture was evaporated to dryness. The residue wascrystallized from ethanol to give 1.8 g of the hydrochloride salt of theamino-hydantoin (10) as a white solid, mp 282°-285° C. (decomp.).

Analysis: Calculated for C₁₉ H₂₁ N₃ O₂. HCl: C, 63.41; H, 6.16; N,11.68. Found: C, 62.62; H, 6.15; N, 11.31.

Mass spectrum (70 eV) m/e: 324 [MH⁺ ], 293 [M⁺ minus CH₂ NH₂ ].

The hydrochloride salt was dissolved in 50 ml of H₂ O and neutralizedwith solid sodium bicarbonate. The free base precipitated and wasrecrystallized from ethanol to give 1.1 g of the amino-hydantoin (10) asa white solid, mp 137°-138° C.

Analysis: Calculated for C₁₉ H₂₁ N₃ O₂ : C, 70.59; H, 6.55; N, 13.00.Found: C, 70.08; H, 6.50; N, 12.55.

N-[4-(5,5-Diphenylhydantoinyl-3)-butyl]-7-β-galactosylcoumarin-3-carboxamide(12)

In a 500 ml flask was placed a solution containing 24 g of potassiumhydroxide dissolved in 80 ml of H₂ O and 240 ml of methanol. Thesolution was cooled to 5° C. and stirred while 20 g (0.035 mols) ofethyl-7-β-galactosylcoumarin-3-carboxylate [Burd et al, Clin. Chem.23:1402 (1977)] was added in one portion. After stirring for 5 minutes,the reaction was heated for 15 hours at 50° C. When cool, the methanolwas removed under reduced pressure. The concentrated aqueous solutionwas acidified to pH 2.0 with concentrated hydrochloric acid. The whiteprecipitate was collected, washed with cold H₂ O and recrystallized fromhot H₂ O. The crystals were washed with acetone and dried at 80° C. for1 hour. This gave 12 g of 7-β-galactosylcoumarin-3-carboxylic acid aswhite crystals, mp 250°-255° C.

Analysis: Calculated for C₁₆ H₁₆ O₁₀ : C, 52.17; H, 4.38. Found: C,52.31; H, 4.63.

A mixture of 737 mg (2.0 mmol) of 7-β-galactosylcoumarin-3-carboxylicacid, 20 ml of dry DMF, and 0.278 ml (202 mg, 2 mmol) of triethylaminewas placed in a 3-necked, 50 ml round bottom flask fitted with astirrer, argon inlet-outlet, and thermometer. When the acid had alldissolved, the contents of the flask were cooled to -10° C. and 273 mg(2 mmol) of isobutyl chloroformate was added. After 10 minutes, 808 mg(2.5 mmol) of 3-(4-aminobutyl)-5,5-diphenylhydantoin (10) and anadditional 0.278 ml of triethylamine was added for reaction with theformed mixed anhydride (11). Thirty minutes later the reaction wasallowed to warm to room temperature and stirred for one hour.

Silica gel 60, 7 g, was added to the reaction mixture and the solventevaporated under high vacuum. The impregnated silica gel was placed atopa column of 200 g of silica gel 60 made up in ethyl acetate. The columnwas eluted with a gradient of 2 liters of ethyl acetate to 2 liters of1:1 (v:v) ethyl acetate:ethanol. Fifteen ml fractions were collected.Fractions 128 to 175 were combined and evaporated to give a gummy solid.Recrystallization from ethanol gave 550 mg of the labeled conjugate (12)as fine white crystals, mp 145° C.

Analysis: Calculated for C₃₅ H₃₅ N₃ O₁₁ : C, 62.40; H, 5.24; N, 6.24.Found: C, 61.72; H, 5.30; N, 6.21.

Mass Spectrum (17 ma) m/e: 674 [MH⁺ ], 512 [MH⁺ minus C₆ H₁₀ O₅ ].

[α]_(D) =-30.48° (c 1.0, methanol).

The above-described synthesis of the N³ -labeled conjugate (12), n=4,can be modified to yield labeled conjugates wherein n=2 through 6 byreplacing the starting material 3-(4-carboxybutyl)-5,5-diphenylhydantoin(9) in the synthesis of the amino-hydantoin (10) with the appropriate3-(ω-carboxyalkyl)-5,5-diphenylhydantoin as follows:

    ______________________________________                                        n                   alkylene                                                  ______________________________________                                        2                   ethylene                                                  3                   propylene                                                 5                   pentylene                                                 6                   hexylene                                                  ______________________________________                                    

2-II. PREPARATION OF THE IMMUNOGEN CONJUGATE

24.3 mg (75 μmol) of 3-(4-carboxybutyl)-5,5-diphenylhydantoin wasdissolved in 0.75 ml dioxane. The solution was cooled to 5° to 10° C. inan ice water bath and 17.5 μl (8.15 mg. 75 μmol) of ethyl chloroformatewas added. The solution was allowed to react for 15 minutes at 5° to 10°C. before adding it to the protein solution. The protein solution wasprepared by dissolving 125 mg (2.08 μmol) of bovine serum albumin (BSA)in 3.25 ml H₂ O containing 125 μl of 1 N sodium hydroxide. While vortexmixing, 3.25 ml dioxane was slowly added to the alkaline BSA solution.The BSA solution was placed in an ice bath and the activateddiphenylhydantoin derivative added. The solution was mixed and allowedto react for 2 hours.

The reaction mixture was brought to room temperature and chromatographedwith 50 mM Tris buffer [tris(hydroxymethyl)aminomethane], pH 8.2,through a column of G-25 Sephadex (2.5×50 cm). The ultravioletabsorbance of the 12 ml fractions was monitored and the immunogenconjugate eluting in the column void volume (fractions 7 to 10) waspooled. The unreacted diphenylhydantoin derivative eluted in fractions15 to 20. Analysis of the absorption spectrum of thediphenylhydantoin-BSA conjugate indicated 28 moles of diphenylhydantoinderivative per mole of BSA.

2-III. BINDING ASSAY FOR DIPHENYLHYDANTOIN

A. Reagents and Apparatus

The reagents and apparatus were the same as those used in the assayemploying the N¹ -derivatives described in 1-III above except that theantiserum was obtained by immunizing rabbits with the N³ -conjugatedimmunogen prepared according to 2-II above.

B. Assay Procedure

The procedure was the same as that used in the assay employing the N¹-derivatives described in 1-III above except that the solutions oflabeled conjugate used were 10 μl aliquots of an 8 μM aqueous solutionof the N³ -labeled conjugate (prepared as described in 2-I above) in0.2% (volume:volume) Tween-20 surfactant.

C. Results

The results, expressed as described in 1-III above, are given in thefollowing table:

    ______________________________________                                        diphenylhydantoin                                                             concentration in      percent of                                              standard (μg/ml)   competition                                             ______________________________________                                        2500                  68.9                                                    250                   60.0                                                    25                    47.2                                                    2.5                   23.2                                                    0.25                  1.1                                                     ______________________________________                                    

The results demonstrate that the labeled conjugate and the antiserumprepared using the immunogen conjugate are useful in an assay fordiphenylhydantoin.

3. o-Phenyl Derivatives 3-III. PREPARATION OF THE LABELED CONJUGATE

The o-phenyl-conjugated β-galactosyl-umbelliferone-diphenylhydantoinlabeled compounds are prepared according to the reaction scheme shown inTable 4. This synthetic route is exemplified by the following method forpreparingN-{4-[2-(5-phenylhydantoinyl-5)-phenoxy]-butyl}-7-β-galactosylcoumarin-3-carboxamide(16). To follow this synthesis in Table 4, n equals 4.

2-[4-(N-Phthalimido)-butoxy]-benzophenone (13).

In a 1 liter, 3-neck round bottom flask was placed 8.64 g of a 50%suspension of sodium hydride (NaH) in mineral oil (0.18 mol). The NaHwas washed free of mineral oil with hexane under an argon atmosphere.The washed NaH was then suspended in 350 ml of dry dimethylformamide(DMF) and stirred while a solution of 34.4 g (0.173 mol) of2-hydroxybenzophenone in 40 ml of DMF was added over a 1 hour period.Thirty minutes after the addition was complete, 52 g (0.184 mol) ofN-(4-bromobutyl)-phthalimide in 150 ml of dry DMF was added over a 20minute period. After stirring at room temperature for 18 hours, thereaction was diluted with 200 ml of water (H₂ O) and the precipitatecollected and dried to yield 49 g of the benzophenone (13), mp 119°-121°C. A 1 g sample was recrystallized from ethanol to give 740 mg of whiteneedles, mp 121°-122° C.

Analysis: Calculated for C₂₅ H₂₁ NO₄ : C, 75.17; H, 5.30; N, 3.51.Found: C, 74.78; H, 5.26; N, 3.79.

NMR Spectrum (CDCl₃): δ 1.5 (m, 4H), 3.5 (m, 2H), 3.9 (m, 2H).

                                      TABLE 4                                     __________________________________________________________________________     ##STR12##                                                                     ##STR13##                                                                    __________________________________________________________________________

5-[2-(4-N-Formylaminobutoxy)-phenyl]-5-phenylhydantoin (14)

A mixture of 22.4 g (0.056 mol) of2-[4-(N-phthalimido)-butoxy]-benzophenone (13), 4.15 g (0.064 mol) ofpotassium cyanide, 17.3 g (0.18 mol) of ammonium carbonate, 24 ml of H₂O, and 200 ml of DMF was placed in a steel autoclave and heated at 110°C. for 4 days. The contents were cooled and adsorbed onto 100 g ofsilica gel 60 and placed atop a 700 g column of silica gel made up in9:1 (v:v) carbon tetrachloride:acetone. Elution was with the samesolvent and fractions of approximately 20 ml volumes were collected.Fractions 276 to 803 were combined and evaporated to give 4.65 g ofsolid. Recrystallization from ethanol gave 2.65 of the hydantoin (14) asa white solid, mp 201°-203° C.

Analysis: Calculated for C₂₀ H₂₁ N₃ O₄ : C, 65.38; H, 5.76; N, 11.44.Found: C, 65.23; H, 5.79; N, 11.47.

NMR Spectrum (NaOD-D₂ O): δ 1.2 (m, 4H), 3.0 (m, 2H), 3.2 to 4.0 (m,2H).

5-[2-(4-Aminobutoxy)-phenyl]-5-phenylhydantoin (15)

A solution of 3.5 g (9.4 mmol) of5-[2-(4-N-formylamino-butoxy)-phenyl]-5-phenylhydantoin (14) in 100 mlof 1 N sodium hydroxide was heated on the steam bath for 24 hours. Thesolution was cooled and nuetralized with carbon dioxide untilprecipitation ceased. The precipitate was filtered and recrystallizedtwice; first from pyridine-2-propanol, then from methanol to give 1.5 gof the aminohydantoin (15) as fine white crystals, mp 235° C.(decomposed).

Analysis: Calculated for C₁₉ H₂₁ N₃ O₃ : C, 67.24; H, 6.24; N, 12.38.Found: C, 67.56; H, 6.29; N, 12.56.

NMR Spectrum (NaOD-D₂ O): δ 0.9 (m, 4H), 2.2 (m, 2H), 3.2 (m, 2H).

N-{4-[2-(5-Phenylhydantoinyl-5)-phenoxy]-butyl}-7-β-galactosylcoumarin-3-carboxamide(16)

A mixture of 808 mg (2 mmol) of the potassium salt of7-β-galactosylcoumarin-3-carboxylic acid [Burd et al, Clin. Chem.23:1402(1977)] and 20 ml of dry DMF was cooled to 0° C. To this mixturewas added 216 mg (2 mmol) of ethyl chloroformate and the reactionstirred for one hour at this temperature to form the mixed anhydride(6). Then 638 mg (2 mmol) of5-[2-(4-aminobutoxy)-phenyl]-5-phenylhydantoin (15), 244 mg of4-dimethylaminopyridine, and 5 ml of dry pyridine were added. Afterstirring for 5 hours, the reaction was stored overnight at 0° C., thenadsorbed onto 7 g of silica gel 60. The impregnated silica gel wasplaced atop a column of 200 g of silica gel 60 and the column elutedwith a gradient of 2 liters of ethyl acetate to 2 liters of 1:1 (v:v)ethyl acetate:ethanol. Ten ml fractions were collected. Fractions 143 to160 were combined to give approximately 200 mg of the labeled conjugate(16) as a glassy solid.

The solid was taken up in methanol and chromatographed on Sephadex LH-20(45 cm by 3.2 cm, Pharmacia Fine Chemicals), eluting with methanol.Seven ml fractions were collected. Fractions 30 to 40 were combined andevaporated to give 100 mg of the desired labeled conjugate (16) as apale, glassy solid.

Analysis: Calculated for C₃₅ H₃₅ N₃ O₁₂.H₂ O: C, 59.40; H, 5.27; N,5.94. Found: C, 59.51; H, 5.04; N, 6.14.

[α]_(D) =-39.04° (c 1.0, methanol).

The above-described synthesis of the o-phenyl-labeled conjugate (16),n=4, can be modified to yield labeled conjugates wherein n=2 through 6by replacing the starting material N-(4-bromobutyl)-phthalimide in thesynthesis of the phthalimide (13) with the appropriateN-(ω-bromoalkyl)-phthalimide as follows:

    ______________________________________                                        n                   alkylene                                                  ______________________________________                                        2                   ethylene                                                  3                   propylene                                                 5                   pentylene                                                 6                   hexylene                                                  ______________________________________                                    

3-II. PREPARATION OF THE IMMUNOGEN CONJUGATE

This synthesis is shown schematically in Table 5 and is exemplified forn=4 as follows:

                  TABLE 5                                                         ______________________________________                                         ##STR14##                                                                    ______________________________________                                    

A mixture of 2.52 g (0.11 gram-atom) of sodium and 400 ml of absoluteethanol was stirred until all of the sodium dissolved. To this was added19.8 g (0.1 mol) of 2-hydroxybenzophenone. After 45 minutes the deepyellow solution was combined with 20.9 g (0.1 mol) ofethyl-5-bromovalerate. The reaction was refluxed for 44 hours, thencooled and diluted with 250 ml of 1 N sodium hydroxide and 250 ml ofether. The ether layer was separated and the aqueous phase saturatedwith sodium chloride, then extracted with four 200 ml portions of ether.The ether extracts were combined, dried over anhydrous magnesiumsulfate, filtered, and evaporated to give 40 g of a yellow oil of2-(4-carbethoxybutoxy)-benzophenone (17).

This ester was not characterized but instead was hydrolyzed by refluxingit 15 hours in a mixture of 500 ml of dioxane and 60 ml of concentratedhydrochloric acid. Evaporation gave a brown oil that was partitionedbetween 200 ml of ether and 200 ml of saturated sodium bicarbonatesolution. The layers were separated and the ether phase extracted with asecond 200 ml portion of sodium bicarbonate solution. The bicarbonateextracts were combined, acidified with hydrochloric acid, and extractedwith three 200 ml portions of ether. The combined ether extracts wereevaporated to give 23 g of a brown oil that was chromatographed on 2000g of silica gel 60 eluting with 19:1 (v:v) carbon tetrachloride:acetone.Twenty ml fractions were collected. Fractions 1271 to 1670 were combinedand evaporated to give 11.1 g of the benzophenone (17). Tworecrystallizations from methylene chloride gave white crystals, mp80°-81° C.

Analysis: Calculated for C₁₈ H₁₈ O₄ : C, 72.46; H, 6.09. Found: C,72.29; H, 6.07.

A mixture of 17.8 g (0.06 mol) of 2-(4-carboxybutoxy)-benzophenone (17),4.15 g (0.064 mol) of potassium cyanide, 17.3 g (0.18 mol) of ammoniumcarbonate, 24 ml of H₂ O, and 200 ml of DMF was placed in a steelautoclave and heated to 110° C. for 5 days. The autoclave was cooled andthe contents dissolved in 800 ml of 10% aqueous sodium hydroxidesolution. It was washed with two 400 ml portions of ether and acidifiedto pH 4.5 with 6 N hydrochloric acid. A precipitate occurred that wasfiltered and dried to give 24.6 g of5-[2-(4-carboxybutoxy)-phenyl]-5-phenylhydantoin (18). Tworecrystallizations from aqueous methanol gave a white solid, mp231°-232° C.

Analysis: Calculated for C₂₀ H₂₀ N₂ O₅ : C, 65.20; H, 5.48; N, 7.61.Found: C, 64.92; H, 5.62; N, 7.90.

NMR Spectrum (C₅ D₅ N): δ 1.2 (m, 2H), 1.6 to 2.0 (m, 4H), 3.2 (m, 2H).

27.6 mg (74 μmol) of the acid (18) was suspended in 1.5 ml dioxane. Thesolution was cooled to 5° to 10° C. in an ice water bath and 17.5 μl(14.5 mg, 75 μmol) of tri-N-butylamine added. After mixing, 6 μl (8.15mg, 75 μmol) of ethyl chloroformate was added and mixed. The solutionwas allowed to react for 15 minutes at 5° to 10° C. before adding it tothe protein solution. The protein solution was prepared by dissolving125 mg (2.08 μmol) of bovine serum albumin (BSA, represented in Table 5as PAA) in 3.25 ml H₂ O containing 125 μl of 1 N sodium hydroxide. Whilevortex mixing, 3.25 ml dioxane was slowly added to the alkaline BSAsolution. The BSA solution was then placed in an ice bath and theactivated diphenylhydantoin derivative added. 0.5 ml of water was added2 minutes after the activated diphenyldantoin derivative in order tokeep the BSA in solution.

After 1.75 hours, the reaction mixture was brought to room temperatureand chromatographed with 50 mM Tris buffer, pH 8.2, through a 2.8×42 cmcolumn of G-25 Sephadex (Pharmacia Fine Chemicals). Fractions of 5.1 mlwere collected and the absorbance at 280 nm monitored. The immunogenconjugate (19) eluted in the void volume and these fractions (13 to 19)were pooled. The unreacted diphenyldantoin eluted in fractions 35 to 47.Analysis of the absorbance of the immunogen conjugate indicated 8.1moles of diphenylhydantoin per mole of BSA.

The above-described synthesis of the o-phenyl-immunogen conjugate (19),n=4, can be modified to yield conjugates wherein n=2 through 6 byreplacing ethyl-5-bromovalerate in the described synthesis of thebenzophenone (17) as follows:

    ______________________________________                                        n               starting material                                             ______________________________________                                        2               ethyl-3-bromopropionate                                       3               ethyl-4-bromobutyrate                                         5               ethyl-6-bromocaproate                                         6               ethyl-7-bromoheptanoate                                       ______________________________________                                    

3-III. BINDING ASSAY FOR DIPHENYLHYDANTOIN

A. Reagents and Apparatus

The reagents and apparatus were the same as those used in the assayemploying the N¹ -derivatives described in 1-III above except that theantiserum was obtained by immunizing rabbits with theo-phenyl-conjugated immunogen prepared according to 3-II above and thatthe diphenylhydantoin standards used were prepared at concentrations of0, 5, 10, 20, and 30 μg/ml in serum.

B. Assay Procedure

3.0 ml aliquots of a reagent were prepared in 50 mM Bicine buffer [pH8.2; N,N-bis-(2-hydroxyethyl)-glycine] in a cuvette to contain 0.018units/ml of β-galactosidase and an amount of antiserum sufficient todecrease the reaction rate to about 10% of that observed in the absenceof antibody in the final reaction. To separate aliquots of the reagentwere added 100 μl of the various standard diphenylhydantoin sera diluted1:50 in 0.5% Tween 20 followed by 100 μl of a 1.05 μM aqueous solutionof the o-phenyl-labeled conjugate (prepared as described in 3-I above).After mixing, the reaction mixtures were incubated 20 minutes at 25° C.and the fluorescence measured for each cuvette.

C. Results

The results, expressed in terms of fluorescence units read from theinstrument, are given in the following table:

    ______________________________________                                        diphenylhydantoin                                                             concentration in     fluorescence                                             standard (μg/ml)  units                                                    ______________________________________                                         0                    72                                                       5                   304                                                      10                   425                                                      20                   595                                                      30                   757                                                      ______________________________________                                    

The results demonstrate that the labeled conjugate and the antiserumprepared using the immunogen conjugate are useful in an assay fordiphenylhydantoin.

What is claimed is:
 1. A compound of the formula: ##STR15## wherein φ isphenyl, n=2 through 6, and R is amino.
 2. The compound of claim 1wherein n=4.
 3. A compound of the formula: ##STR16## wherein φ isphenyl, n=2 through 6, and R is amino or carboxyl.
 4. The compound ofclaim 3 wherein n=4.
 5. The compound of claim 3 or 4 wherein R is amino.6. The compound of claim 3 or 4 wherein R is carboxyl.